Method of treating inflammatory arthropathies with suppressors of CpG oligonucleotides

ABSTRACT

The present disclosure relates to oligodeoxynucleotides that suppress an immune response. Methods are disclosed for preventing or treating inflammatory arthropathies by administering a therapeutically effective amount of a suppressive oligodeoxynucleotide.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 10/523,273,filed on Jan. 31, 2005 now U.S. Pat. No. 7,514,415, which is the §371U.S. National Stage of International Application No. PCT/US2003/024205,filed Jul. 31, 2003, which was published in English under PCT Article21(2), which in turn claims the benefit of U.S. Provisional PatentApplication No. 60/400,826, filed Aug. 1, 2002, and to U.S. ProvisionalPatent Application No. 60/401,631, filed Aug. 6, 2002, all of which areincorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to oligodeoxynucleotides that suppress animmune response to CpG oligodeoxynucleotides, and to the use of thesesuppressors in preventing or treating inflammatory arthropathies.

BACKGROUND

Arthritis, an inflammatory disease that affects the synovial membranesof one or more joints in the body, is the most common type of jointdisease. Billions of dollars are spent annually for the treatment ofarthritis and for lost days of work associated with the disease. Thedisease is usually oligoarticular (affects few joints), but may begeneralized. The joints commonly involved include the hips, knees, lowerlumbar and cervical vertebrae, proximal and distal interphangeal jointsof the fingers, first carpometacarpal joints, and first tarsometatarsaljoints of the feet.

One type of arthritis is reactive arthritis, which is an acutenonpurulent arthritis secondary to a urinary tract or gastrointestinalinfection with a variety of microorganisms, including Chlamydiatrachomatis, Yersinia, Salmonella, Shigella, and Campylobacter.Microbial components (and not live organisms) are found in the affectedjoints. The arthritis appears abruptly and tends to involve the kneesand ankles, but sometimes involves the wrists, fingers, and/or toes.Untreated, the arthritis lasts for about a year, then generally abatesand only rarely is accompanied by ankylosing spondylitis. Despiteevidence of disease being triggered by bacterial infection, viablebacteria are rarely present in affected joints and antibiotic treatmentseldom provides relief.

Up to 16% of subjects with gastrointestinal (GI) infection by Salmonellaor Shigella subsequently develop arthritis. Despite this temporalassociation, it is unclear whether live bacteria reaching the affectedjoint are the cause of this arthritis. To date, success in culturingviable microorganisms from the affect joints has been quite limited, andantibiotic treatment rarely is of benefit. Symptomatic treatment isoften accomplished with high doses of non-steroidal anti-inflammatoryagents. In addition, intra-articluar steroid injections are of use.However, a need remains for additional therapies for this disease.

Rheumatoid Arthritis (RA) is a chronic, systemic, inflammatory diseasethat affects the synovial membranes of multiple joints. RA considered anacquired autoimmune disease, and genetic factors appear to play a rolein its development. In most cases of RA, the subject has remissions andexacerbations of the symptoms. Rarely does the disease resolvecompletely, although at times the symptoms might temporarily remit.

Symptomatic medications, such as non-steroidal anti-inflammatory agentsand aspirin, analgesics, and glucocorticoids, are used in the treatmentof rheumatoid arthritis to help reduce joint pain, stiffness andswelling. In addition, low doses of methotrexate, leflunomide,D-Penicillamine, sulfasalazine, gold therapy, minocycline, azathioprine,hydroxychloroquine (and other anti-malarials), and cyclosporine are usedto modify the progression of the disease. However, a need still remainsfor other agents that can be used to alter the progression, orameliorate the symptoms, of this disease.

In view of the above, there exists a need for new therapies to treatinflammatory arthropathies, particularly agents that suppress theinflammation associated with arthritis

BRIEF SUMMARY OF SPECIFIC EMBODIMENTS

Disclosed herein are oligodeoxynucleotides that can be used to suppressimmune activation. These suppressive oligodeoxynucleotides are of use inpreventing and/or treating inflammatory arthropathies, such as, but notlimited to, reactive arthritis and rheumatoid arthritis. The suppressiveoligodeoxynucleotides can be administered locally or systemically. Onespecific, non-limiting example of local administration is byintra-articular injection.

A substantially pure or isolated oligodeoxynucleotide (ODN) is disclosedherein that is at least about 8 nucleotides in length, forms a G tetrad,has a CD value of greater than 2.9, has at least two guanosines, andsuppresses an immune response. Optionally, the suppressive ODN hasmultiple guanosine-rich sequences, and in some examples, the ODN has oneor more TTAGGG motifs. Furthermore, in particular embodiments, the ODNis modified to prevent degradation or is part of an oligodeoxynucleotidedelivery complex that includes a targeting moiety. In one specific,non-limiting example the suppressive ODN suppresses CpG-DNA-inducedimmune activation. Also disclosed herein is a pharmacologicalcomposition that includes the suppressive ODN and a pharmacologicallyacceptable carrier.

A method is disclosed herein for treating or preventing an inflammatoryarthropathy in a subject. The method includes administering atherapeutically effective amount of the suppressive ODN to a subjecthaving or at risk of developing an inflammatory arthropathy, therebytreating or preventing the inflammatory arthropathy.

In another embodiment, a method of treating or preventing aninflammatory arthropathy in a subject that includes contacting immunecells with the suppressive ODN in vitro. The immune cells are andtransferred to a subject having or at risk of developing an inflammatoryarthropathy.

Also described herein is a kit for treating or preventing inflammatoryarthropathies in a subject that includes the suppressive ODN andinstructions for administering the ODN to a subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of diagrams of the structure of a G-tetrad. FIG. 1A is adiagram of the structure of an individual G-tetrad that shows theHoogsteen base pairing. M⁺ represents a monovalent cation such as K⁺ orNa⁺ and dR is the sugar-phosphate backbone. FIG. 1B is a schematicrepresentation showing the possible folded intramolecular quadruplexstructure. FIG. 1C is a schematic showing the GG-base pair formed bymeans of Hoogsteen hydrogen bonds. FIG. 1D is a schematic of anintramolecular hairpin.

FIG. 2 is a set of graphs showing the effect of CpG ODN and suppressiveODN injection into the knee. FIG. 2A is a graph of joint swelling inmice treated with CpG ODN (solid circle), CpG plus control ODN (solidsquare), CpG plus suppressive ODN (open diamond), control ODN (opentriangle), suppressive ODN (inverted open triangle), or PBS (opensquare, N=8-11 mice/group). FIG. 2B is a graph showing histologicchanges in the injected knees. Knees were evaluated four days aftertreatment by a blinded investigator. Scale: 0; no inflammation, 1;sparse, localized perivascular infiltrate, 2; moderate infiltrate, 3;moderate—dense infiltrate with synovial hyperplasia. FIG. 2C is a graphshowing that suppressive ODNs suppress joint swelling. At T=0, bothknees were injected with 25 μg of CpG ODN. The right knee was theninjected with PBS and the left knee with 25 μg of suppressive ODN either0 (solid circles, N=3), 24 (solid squares, N=6) or 48 (solid triangles,N=6) hours later. Results show the difference in swelling between thetwo knees. Statistical significance was assessed by repeated-measuresANOVA using the Proc Mixed procedure (FIG. 2A, 2C) and Wilcoxon Rank sumtest (B). The asterisk (*) indicates that p<0.05.

FIG. 3 is a set of digital images of a photomicrograph showing theeffects of CpG and ODN injection on knee histology in mice. BALB/c kneeswere injected with PBS (FIG. 3A, B), CpG ODN (FIG. 3C, D), CpG pluscontrol ODN (FIG. 3E, F) or CpG plus suppressive ODN (25 μg of each ODN,FIG. 3G, H). Typical histology four days after injection of 25 μg ofeach ODN is shown. Note the cellular infiltrates, perivascularaccumulation of mononuclear cells, and hyperplasia of the synoviallining in the knees of mice injected with CpG ODN. FIGS. 3A, 3C, 3E and3G show 100× magnification; FIGS. 3B, 3D, 3F, and 3H show 400×magnification.

FIG. 4 is a set of graphs that demonstrate that administration ofsuppressive ODN decreases TNFα upregulation following CpG ODN injection.FIG. 4A is a graph of TNFα levels following treatment with ODN. BALB/cspleen cells were stimulated in vitro for 72 hours with 1 μM of variousODN. FIG. 4B is a graph of percent tumor necrosis factor alpha (TNFα)production versus suppressive ODN concentration. RAW 264.7 cells(106/well) were stimulated with 1 μM CpG plus increasing amounts ofsuppressive ODN. The concentration of TNF in culture supernatants after24 hours was measured by ELISA. Data represent the mean+SEM of 5independently studied animals/group. Statistical significance wasassessed by Wilcoxon Rank sum test. FIG. 4C is a digital image of anagarose gel. Joints injected with 25 μg of ODN were processed into RNA 3days later. Representative examples of local TNF and β-actin mRNA levelsare shown. FIG. 4D is a graph of relative TNFα mRNA expression followingtreatment with CpG ODN or CpG ODN and suppressive ODN. Relativeintensity of TNFα vs β-actin mRNA (N=3). The asterisk indicates thatp<0.05.

FIG. 5 is a graph showing that the local administration of suppressiveODNs reduces the pro-inflammatory effect of CpG ODN administration.Optimal suppression of CpG ODN-mediated joint inflammation is attainedwhen the suppressive ODN are administered three days before theinflammatory challenge. Shown is the mean and standard error ofmeasurement (SEM) of 10-11 mice/group pre-treated locally(intra-articularly) with suppressive ODN three days prior to CpG ODNchallenge (open circles), suppressive ODN administered one day prior toCpG ODN challenge (open, inverted triangles), suppressive ODNadministered at the time of the CpG ODN challenge (open triangles). Thejoint swelling of mouse knees injected with control ODN (black diamonds)or phosphate buffered saline (PBS; black circles) three days before theCpG serve as controls.

FIG. 6 is a graph showing that naive mice (and mice pre-treated withcontrol ODN or PBS) developed severe arthritis following local CpG ODNchallenge. In contrast, systemic administration of suppressive ODN threedays prior to local CpG DNA challenge reduced joint swelling andinflammation by 80-85% (p<0.029). BALB/c mice were treated IP with 300μg of suppressive ODN 0-3 days prior to the intra-articular injection of25 μg CpG DNA. Consistent with previous studies, naive mice (and micepre-treated with control ODN or PBS) developed severe arthritisfollowing local CpG ODN challenge.

FIG. 7 is a pair of graphs showing that systemic administration ofsuppressive ODN three days prior to local CpG DNA challenge reducedjoint swelling and inflammation by 80-85% (p<0.029).

FIGS. 8A and 8B are a pair of graphs showing that systemicallyadministered suppressive ODN elicit a population of regulatory cellsthat inhibit CpG-induced arthritis. As expected, spleen cells fromuntreated donors had no effect on the development of CpG-inducedarthritis. By comparison, the transfer of 20×10⁶ spleen cells fromsuppressive ODN treated donors significantly reduced joint swelling andinflammation in the recipients.

FIG. 9 is a graph showing that CD11c⁺ cells are responsible for theresistance to CpG-induced arthritis. Magnetic beads were used to depleteor enrich specific cell subpopulations from donor spleens. Depletion ofCD19⁺ B cells, T cells, or NK cells had no effect on CpG-inducedarthritis. However, removal of CD11c⁺ dendritic cells resulted in acomplete abrogation of the suppressive activity of the transferredspleen cell population. Similarly, the transfer of only 5×10⁵ CD11c⁺enriched cells from suppressive ODN treated mice to normal recipientsconferred resistance to CpG-induced arthritis.

FIGS. 10A through 10F are graphs showing the factors contributing to thesuppression of CpG-induced immune activation. FIG. 10A is a graphshowing that mammalian DNA suppresses CpG DNA-induced immune activation.FIG. 10B is a graph showing that the telomeric TTAGGG repetitive motifis suppressive. SEQ ID NOs: 2, 3, 4, 113 and 114 are shown. FIG. 10C isa graph showing that the suppressive motif is active in trans and cisconformations. FIG. 10D is a graph showing that suppressive ODNsselectively block CpG DNA-induced immune activation. FIG. 10E is a graphshowing that Poly Gs are critical for suppression (SEQ ID NOs: 114-122are shown). FIG. 10F is a graph showing that G-tetrad formingnon-telomere sequences are also suppressive (SEQ ID NOs: 12 and SEQ IDNOs: 123-126 are shown).

FIG. 11 is a pair of graphs showing that G-tetrad-forming suppressiveODNs selectively suppress CpG-induced immune activation. FIG. 11A is agraph showing that oligonucleotides (ODNs) containing suppressive motifsinhibit CpG-induced immune activation in a dose dependent fashion. FIG.11B is a graph showing G-tetrad formation and suppressive activity ofphosphorothioate and 7-deaza guanosine (DG) modified ODNs.

FIG. 12 is a graph showing the effect of suppressive ODN on CpG DNA andConcavalin A induced IFNγ production. BALB/c spleen cells werestimulated with 1 μM CpG ODN(ODN1555, ODN1466), 50 μg/ml of bacterialDNA, or 5 μg/ml Con A. The response of these cultures was compared tocells co-stimulated with 1 μM of control ODN1612, suppressive ODN1502 orsuppressive ODNH154. The number of IFNγ secreting cells was determinedby ELIspot after 18 hours. Data represent the average+standard deviation(SD) of triplicate cultures. The experiment was repeated three timeswith similar results.

FIG. 13 is a graph showing the concentration effects of suppressive ODN.BALB/c spleen cells were stimulated with 1 μM CpG ODN1555 or ODN1466plus increasing amounts of suppressive ODN1502 or ODNH154. Cytokinelevels in culture supernatants were measured by Enzyme-LinkedImmunosorbent Assay (ELISA) after 24 hours. Results represent themean+SD of 4 different experiments.

FIG. 14 is a graph showing the kinetics of suppressive ODN. BALB/cspleen cells were stimulated with 1 μM CpG ODN1555. At various times, 1μM suppressive ODN1502 was added. Cytokine levels in culturesupernatants were measured by ELISA after 24 hours. Results representthe mean of two independent experiments.

FIG. 15 is a graph showing the effect of removing CpG ODN from culturedcells. 1 μM of CpG ODN1555 was added to BALB/c spleen cells at time(T)=0. The cells were washed free of this ODN after various incubationperiods. IFNγ and IL-12 levels in culture supernatants were measured byELISA after 24 hours. Results represent the average+SD of duplicatecultures. Similar results were obtained in studies of CpG ODN1466.

FIG. 16 is a graph showing that suppressive ODN do not block the bindingor uptake of CpG ODN. BALB/c spleen cells were incubated with 1 μM ofCpG ODN1555 (black bar) plus 1 μM of suppressive ODN1502 (grey bar) orcontrol ODN1612 (white bar). The percent of cells that bound orinternalized the CpG ODN was determined by FACS. Similar results wereobtained using CpG ODN1466, suppressive ODNH154 and control ODN1471.

FIG. 17 is a schematic diagram of the timing of type II collageninjection and ODN administration for DBA/1 LacJ mice. Ten to twentyDBA/1 LacJ mice per group were injected with type II collagen incomplete Freund's adjuvant (CII/CFA) on day 0, and with type II collagenin incomplete Freund's adjuvant (CII/IFA) on day 21 to induce arthritis.The study groups included animals treated with 200 μg of suppressive ODNA151 (SEQ ID NO: 2), control ODN 1612 (SEQ ID NO: 29) or PBS on days −3,0, 3, 7, 10, 14, 18 and 21. The incidence of arthritis and clinicalscore were monitored twice weekly. Antigen-specific humoral and cellularimmune responses, and local expression of pro-inflammatory cytokines,were also investigated.

FIG. 18 is a pair of graphs showing that treatment with ODN A151 (SEQ IDNO: 2) significantly reduced both the percentage of mice that developedarthritis (FIG. 18A), and the arthritis clinical score (FIG. 18B).Standard curves were constructed by serial dilutions of a mixture ofsera from arthritic mice. A single asterisk (*) indicates that p<0.05; adouble asterisk (**) indicates that p<0.01, as compared with PBS-controlmice.

FIG. 19 is a graph showing that production of anti-type 2 collagen (CII)antibody is suppressed by A151 (SEQ ID NO: 2) treatment. Sera werecollected on day 35 and assayed for IFNγ by ELISA assay, as describedabove. Standard curves were constructed by serial dilutions of a mixtureof sera from arthritic mice. A single asterisk (*) indicates thatp<0.05; a double asterisk (**) indicates that p<0.01, as compared withPBS-control mice.

FIG. 20 is a pair of graphs showing that production of anti-CII antibodyis suppressed by A151 treatment. Spleen cells from naive, PBS orA151-treated mice were isolated on day 22 (FIG. 20A) or 35 (FIG. 20B).Cells were stimulated in vitro with 50 μg/ml of CII for 72 hours andculture supernatants were assayed for IFN-gamma detection. Each grouprepresents results from six mice (two separate experiments). A singleasterisk (*) indicates that p<0.05; a double asterisk (**) indicatesthat p<0.01, as compared with naive or PBS-control mice.

FIG. 21 is a digital image of a gel showing that in vivo (local)expression of proinflammatory cytokine is also suppressed by treatmentwith ODN A151. The hind paws of treated animals were removed on day 35.Total RNA was extracted from tissue homogenates, and mRNA of thepro-inflammatory cytokine IL-1 β was monitored by RT-PCR. Each groupcontains three mice.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs 1-25 are suppressive ODN sequences.

SEQ ID NOs 28, 29, and 31 are control ODN sequences.

SEQ ID NOs 26, 27, and 30 are immunostimulatory CpG sequences.

SEQ ID NOs 32-33 are TNFα primers.

SEQ ID NOs 34-35 are β-actin primers.

SEQ ID NOs 113-126 are ODN sequences.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Abbreviations

A: adenine

Ab: antibody

C: cytosine

CD: circular dichroism

CII: type II collagen

CII/CFA: type II collagen in complete Freund's adjuvant

CII/IFA: type II collagen in incomplete Freund's adjuvant

CpG ODN: an oligodeoxynucleotide including a CpG motif.

DC: dendritic cell

DG: deaza guanosine

ELISA: Enzyme-Linked Immunosorbent Assay

FCS: fetal calf serum

G: guanine

GI: gastrointestinal

GU: genitourinary

h: hour

IFN-α interferon alpha

IFN-γ: interferon gamma

IL-10: interleukin 10

LPS: lipopolysaccharide

μg: microgram

mm: millimeter

mRNA: messenger ribonucleic acid.

ODN: oligodeoxynucleotide

PBS: phosphate buffered saline

Pu: purine

Py: pyrimidine

s.c.: subcutaneous

SD: standard deviation

SE: standard error

T: thymine

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

5′ and 3′: DNA molecules are said to have “5′ ends” and “3′ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotide is referred to as the“5′end” if its 5′ phosphate is not linked to the 3′oxygen of amononucleotide pentose ring. An end of an oligonucleotide is referred toas the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate ofanother mononucleotide pentose ring. As used herein, a nucleic acidsequence, even if internal to a larger oligonucleotide, also may be saidto have 5′ and 3′ ends. In either a linear or circular DNA molecule,discrete elements are referred to as being “upstream” or 5′ of the“downstream” or 3′ elements. This terminology reflects thattranscription proceeds in a 5′ to 3′ direction along the DNA strand. Thepromoter and enhancer elements which direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Anti-inflammatory or anti-arthritis agent: A medication that, as well ashaving pain-relieving (analgesic) effects, has the effect of reducinginflammation when used over a period of time. Anti-inflammatory oranti-arthritis agents include both steroids and non-steroidalanti-inflammatory agents. Anti-inflammatory or anti-arthritis agentsinclude, but are not limited to cortisone, prednisone, celecoxib,choline magnesium trisalicylate, diclofenac, diclofenac potassium,diclofenac XR, diflunisal, etodolac, etodolac ER, fenoprofen,flurbiprofen oral, ibuprofen, indomethacin, indomethacin SR,indomethacin suppositories, ketoprofen, ketoprofen ER, meclofenamate,meloxicam, nabumetone, naproxen, naproxen CR, naproxen ER, oxaprozin,piroxicam, rofecoxib, salsalate, sulindac, and tolmetin sodium.

Antigen: A compound, composition, or substance that can stimulate animmune response such as the production of antibodies or a T-cellresponse in an animal, including compositions that are injected orabsorbed into an animal. An antigen reacts with the products of specifichumoral or cellular immunity, including those induced by heterologousimmunogens. The term “antigen” includes all related antigenic epitopes.

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′→3′ strand, referred to as the plus strand, and a 3′→5′strand (the reverse compliment), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′→3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a dsDNAtarget. In one embodiment, an antisense molecule specifically hybridizesto a target mRNA and inhibits transcription of the target mRNA.

Arthritis: Arthritis is an inflammatory disease that affects thesynovial membranes of one or more joints in the body. It is the mostcommon type of joint disease, and it is characterized by theinflammation of the joint. The disease is usually oligoarticular(affects few joints), but may be generalized. The joints commonlyinvolved include the hips, knees, lower lumbar and cervical vertebrae,proximal and distal interphangeal joints of the fingers, firstcarpometacarpal joints, and first tarsometatarsal joints of the feet.

One type of arthritis is reactive arthritis, which is an acutenonpurulent arthritis secondary to a urinary tract or gastrointestinalinfection with a variety of microorganisms, including Chlamydiatrachomatis, Yersinia, Salmonella, Shigella, and Campylobacter.Microbial components are found in the affected joints. The arthritisappears abruptly and tends to involve the knees and ankles, butsometimes involves the wrists, fingers, and/or toes. Untreated, thearthritis lasts for about a year, then generally abates and only rarelyis accompanied by ankylosing spondylitis. Despite evidence of diseasebeing triggered by bacterial infection, viable bacteria are rarelypresent in affected joints and antibiotic treatment seldom providesrelief.

Another type of arthritis is rheumatoid arthritis. Rheumatoid arthritisis a chronic, systemic, inflammatory disease that affects the synovialmembranes of multiple joints in the body. Because the disease issystemic, there are many extra-articular features of the disease aswell. For example, neuropathy, scleritis, lymphadenopathy, pericarditis,splenomegaly, arteritis, and rheumatoid nodules are frequent componentsof the disease. In most cases of rheumatoid arthritis, the subject hasremissions and exacerbations of the symptoms. Rheumatoid arthritisconsidered an autoimmune disease that is acquired and in which geneticfactors appear to play a role.

Autoimmune disorder: A disorder in which the immune system produces animmune response (for example, a B cell or a T cell response) against anendogenous antigen, with consequent injury to tissues. For example,rheumatoid arthritis is an autoimmune disorder, as are Hashimoto'sthyroiditis, pernicious anemia, Addison's disease, type I diabetes,systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome,dermatomyositis, lupus erythematosus, multiple sclerosis, myastheniagravis, Reiter's syndrome, and Grave's disease, among others.

CD value: The formation of G-tetrads yields a complex with differentphysical properties than the individual oligonucleotides.Spectroscopically, this is manifested by an increase in circulardicroism (CD), and an increase in peak absorbance to the 260-280 nmwavelength owing to the formation of secondary structures. Thus, aconvenient method for identifying oligonucleotides that form G-tetradsis to study their CD values. An increase in peak ellipticity values togreater than 2.0 is typical of a G-tetrad forming oligonucleotide. Thehigher the ellipticity value, the greater the tetrad-forming capacity ofthe oligonucleotide.

CpG or CpG motif: A nucleic acid having a cytosine followed by a guaninelinked by a phosphate bond in which the pyrimidine ring of the cytosineis unmethylated. The term “methylated CpG” refers to the methylation ofthe cytosine on the pyrimidine ring, usually occurring the 5-position ofthe pyrimidine ring. A CpG motif is a pattern of bases that include anunmethylated central CpG surrounded by at least one base flanking (onthe 3′ and the 5′ side of) the central CpG. Without being bound bytheory, the bases flanking the CpG confer part of the activity to theCpG oligodeoxynucleotide. A CpG oligonucleotide is an oligonucleotidethat is at least about ten nucleotides in length and includes anunmethylated CpG. CpG oligonucleotides include both D and K typeoligodeoxynucleotides (see below). CpG oligodeoxynucleotides aresingle-stranded. The entire CpG oligodeoxynucleotide can be unmethylatedor portions may be unmethylated. In one embodiment, at least the C ofthe 5′ CpG 3′ is unmethylated.

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking.

D Type Oligodeoxynucleotide (D ODN): An oligodeoxynucleotide includingan unmethylated CpG motif that has a sequence represented by theformula:

5′ RY-CpG-RY 3′ (SEQ ID NO: 36)

wherein the central CpG motif is unmethylated, R is A or G (a purine),and Y is C or T (a pyrimidine). D-type oligodeoxynucleotides include anunmethylated CpG dinucleotide. Inversion, replacement or methylation ofthe CpG reduces or abrogates the activity of the D oligodeoxynucleotide.

In one embodiment, a D type ODN is at least about 16 nucleotides inlength and includes a sequence represented by Formula III of PCTApplication No. PCT/US02/30532, which is incorporated by referenceherein. The oligonucleotides have a sequence set forth as:

(SEQ ID NO: 36-112) 5′ X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄ X₄X₅X₆(W)_(M)(G)_(N)-3′

wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10.Generally D ODNs can stimulate a cellular response. For example, D ODNsstimulate natural killer cells and the maturation of dendritic cells.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, for example, thatelicit a specific immune response. An antibody binds a particularantigenic epitope.

Functionally Equivalent: Sequence alterations, for example in asuppressive ODN, that yield the same results as described herein. Suchsequence alterations can include, but are not limited to, deletions,base modifications, mutations, labeling, and insertions.

G-tetrad: G-tetrads are G-rich DNA segments that can accommodate complexsecondary and/or tertiary structures (see FIG. 1). A G-tetrad involvesthe planar association of four Gs in a cyclic Hoogsteen hydrogen bondingarrangement (this involves non-Watson Crick base-pairing). In general,either a run of two or more contiguous Gs or a hexameric region inwhich >50% of the bases are Gs, is needed for an ODN to form a G-tetrad.The longer the run of contiguous Gs, and the higher the G content of theODN, the higher the likelihood of G-tetrad formation, as reflected byhigher CD or ellipticity values.

Oligonucleotides that form G-tetrads can also form higher-levelaggregates that are more easily recognized and taken up by immune cells,for example, through scavenger receptors or by nucleolin.

Guanosine-rich sequence: A hexameric region of a nucleotide sequence inwhich >50% of the bases are Gs.

Immediately adjacent: Two nucleic acid sequences are “immediatelyadjacent” if a spacer of 0-9 nucleotides is included between the nucleicacid sequences. In one example, one nucleic acid sequence includes animmunostimulatory CpG motif and the other nucleic acid sequence is asuppressive nucleic acid sequence. Thus an immunostimulatory CpG motifand the suppressive ODN sequence are immediately adjacent if they areseparated by less than 9 nucleotides.

Immunostimulatory CpG ODN: An oligodeoxynucleotide, which contains acytosine, guanine dinucleotide sequence and stimulates (for example, hasa mitogenic effect) vertebrate immune cells. The cytosine, guanine isunmethylated. Both D and K type CpG ODNs are immunostimulatory (see inVerthelyi et al., J. Immunol. 166:2372-2377, 2001, which is hereinincorporated by reference).

Immunosuppressive agent: A molecule, such as a chemical compound, smallmolecule, steroid, nucleic acid molecule, or other biological agent,that can decrease an immune response such as an inflammatory reaction.Immunosuppressive agents include, but are not limited to an agent of usein treating arthritis (anti-arthritis agent). Specific, non-limitingexamples of immunosuppressive agents are non-steroidal anti-inflammatoryagents, cyclosporine A, FK506, and anti-CD4. In additional examples, theagent is a biological response modifier, such as KINERET® (anakinra),ENBREL® (etanercept), or REMICADE® (infliximab), a disease-modifyingantirheumatic drug (DMARD), such as ARAVA® (leflunomide), a steroid,such as prednisone or cortisone, a nonsteroidal anti-inflammatory drug(NSAID), specifically a Cyclo-Oxygenase-2 (COX-2) inhibitor, such asCELEBREX® (celecoxib) and VIOXX® (rofecoxib), or another product, suchas HYALGAN® (hyaluronan) and SYNVISC® (hylan G-F20).

Immune response: A response of a cell of the immune system, such as a Bcell or T cell to a stimulus. In one embodiment, the response isspecific for a particular antigen (an “antigen-specific response”).

Infectious agent: An agent that can infect a subject, including, but notlimited to, viruses, bacteria, and fungi.

Examples of infectious virus include: Retroviridae (for example, humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (for example, polio viruses, hepatitis A virus;enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (such as strains that cause gastroenteritis); Togaviridae(for example, equine encephalitis viruses, rubella viruses); Flaviridae(for example, dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (forexample, vesicular stomatitis viruses, rabies viruses); Filoviridae (forexample, ebola viruses); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (for example, influenza viruses); Bungaviridae (forexample, Hantaan viruses, bunga viruses, phleboviruses and Nairoviruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (forexample, reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2,varicella zoster virus, cytomegalovirus (CMV), herpes viruses);Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (such as African swine fever virus); and unclassifiedviruses (for example, the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (for example, Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli.

Examples of infectious fungi include, but are not limited to,Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

Other infectious organisms (such as protists) include: Plasmodiumfalciparum and Toxoplasma gondii.

Inflammatory arthropathy: An inflammatory arthropathy is an inflammatorydisease affecting one or more joints, such as an inflammatory diseasethat affects the synovial membranes of one or more joints. Inflammatoryarthropathies include, for example, arthritis, ankylosing spondylitis,Reiter's syndrome, psoriatic arthropathy, enteropathis spondylitis,juvenile arthropathy, and reactive arthropathy.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, for example, otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

K Type Oligodeoxynucleotide (K ODN): An oligodeoxynucleotide includingan unmethylated CpG motif that has a sequence represented by theformula:

5′ N₁N₂N₃Q-CpG-WN₄N₅N₆ 3′

wherein the central CpG motif is unmethylated, Q is T, G or A, W is A orT, and N₁, N₂, N₃, N₄, N₅, and N₆ are any nucleotides. In oneembodiment, Q is a T. Generally K ODNs can stimulate a humoral response.For example, K ODNs stimulate the production of IgM. K type CpG ODNshave been previously described (see U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6239,116, 6,339,068; 6,406,705, and 6,429,199,which are herein incorporated by reference).

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cell, subdivided between 2 main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases.

Maturation: The process in which an immature cell, such as dendriticcell, changes in form or function to become a functional mature cell,such as an antigen presenting cell (APC).

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide or oligo: Multiple nucleotides (for example, moleculescomprising a sugar, for example, ribose or deoxyribose) linked to aphosphate group and to an exchangeable organic base, which is either asubstituted pyrimidine (Py) (for example, cytosine (C), thymine (T) oruracil (U)) or a substituted purine (Pu) (for example, adenine (A) orguanine (G)). The term “oligonucleotide” as used herein refers to botholigoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs). Theterm “oligonucleotide” also includes oligonucleosides (anoligonucleotide minus the phosphate) and any other organic base polymer.Oligonucleotides can be obtained from existing nucleic acid sources (forexample, genomic or cDNA), but are preferably synthetic (for example,produced by oligonucleotide synthesis).

A “stabilized oligonucleotide” is an oligonucleotide that is relativelyresistant to in vivo degradation (for example via an exo- orendo-nuclease). In one embodiment, a stabilized oligonucleotide has amodified phosphate backbone. One specific, non-limiting example of astabilized oligonucleotide has a phosphorothioate modified phosphatebackbone (wherein at least one of the phosphate oxygens is replaced bysulfur). Other stabilized oligonucleotides include: nonionic DNAanalogs, such as alkyl- and aryl-phosphonates (in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group), phophodiesterand alkylphosphotriesters, in which the charged oxygen moiety isalkylated. Oligonucleotides which contain a diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

An “immunostimulatory oligonucleotide,” “immunostimulatory CpGcontaining oligodeoxynucleotide,” or “CpG ODN,” refers to anoligodeoxynucleotide, which contains a cytosine, guanine dinucleotidesequence and stimulates (for example, has a mitogenic effect) vertebrateimmune cells. The cytosine, guanine is unmethylated.

An “oligonucleotide delivery complex” is an oligonucleotide associatedwith (for example, ionically or covalently bound to; or encapsulatedwithin) a targeting means (for example, a molecule that results in ahigher affinity binding to a target cell (for example, B-cell or naturalkiller (NK) cell) surface and/or increased cellular uptake by targetcells). Examples of oligonucleotide delivery complexes includeoligonucleotides associated with: a sterol (for example, cholesterol), alipid (for example, cationic lipid, virosome or liposome), or a targetcell specific binding agent (for example, a ligand recognized by atarget cell specific receptor). Preferred complexes must be sufficientlystable in vivo to prevent significant uncoupling prior tointernalization by the target cell. However, the complex should becleavable or otherwise accessible under appropriate conditions withinthe cell so that the oligonucleotide is functional. (Gursel, J. Immunol.167: 3324, 2001)

Parenteral: Administered outside of the intestine, for example, not viathe alimentary tract. Generally, parenteral formulations are those thatwill be administered through any possible mode except ingestion. Thisterm especially refers to injections, whether administeredintravenously, intrathecally, intramuscularly, intraperitoneally,intra-articularly, or subcutaneously, and various surface applicationsincluding intranasal, intradermal, and topical application.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. Pharmaceutical agents include, but are notlimited to, chemotherapeutic agents and anti-infective agents.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to have a predisposition to a disease such as an autoimmunedisorder. An example of a person with a known predisposition is someonewith a history of diabetes in the family, or who has been exposed tofactors that predispose the subject to a condition, such as lupus orarthritis. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell.Preferably, a preparation is purified such that the protein or peptiderepresents at least 50% of the total peptide or protein content of thepreparation.

Suppressive ODN: DNA molecules of at least eight nucleotides in length,wherein the oligodeoxynucleotide forms a G-tetrad, and has a CD value ofgreater than about 2.9. In a suppressive ODN the number of guanosines isat least two. In one embodiment, a suppressive ODN inhibits immuneactivation caused by CpG DNA when administered prior to, concurrentlywith, or after the administration of a CpG ODN at least about 8nucleotides in length.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents.

Therapeutically effective amount of [a compound]: A quantity of aspecified compound sufficient to achieve a desired effect in a subjectbeing treated. For instance, this can be the amount of a suppressive ODNnecessary to suppress CpG-induced immune cell activation in a subject,or a dose sufficient to prevent advancement, or to cause regression of adisease, or which is capable of relieving symptoms caused by a disease,such as pain or swelling.

The therapeutically effective amount of a suppressive ODN can beadministered systemically or locally. In addition, a therapeuticallyeffective amount of a suppressive ODN can be administered in a singledose, or in several doses, for example daily, during a course oftreatment. However, the therapeutically effective amount of the ODN willbe dependent on the preparation applied, the subject being treated, theseverity and type of the affliction, and the manner of administration ofthe compound. For example, a therapeutically effective amount of asuppressive ODN can vary from about 0.01 mg/kg body weight to about 1g/kg body weight in some embodiments, or from about 0.01 mg/kg to about60 mg/kg of body weight, based on efficacy.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease, such as pain or swelling.

III. Description of Several Embodiments

A. Suppressive Oligodeoxynucleotides and Guanosine-Quadruplexes(G-Tetrads)

The present disclosure relates to a class of DNA motifs that selectivelyinhibits or suppresses immune activation. Optimal activity is observedusing multimers of these motifs, which are rich in G bases and capableof forming G-quadruplexes (G-tetrads). G-tetrads are G-rich DNA segmentsthat can accommodate complex secondary and/or tertiary structures (seeFIG. 1). The suppressive ODNs of the disclosure are highly specific (forexample, are neither toxic nor non-specifically immunosuppressive), andare useful for inhibiting an immune response. In one embodiment, asuppressive ODN is of use for blocking an immune response, such as anautoimmune response.

A G-tetrad involves the planar association of four Gs in a cyclicHoogsteen hydrogen bonding arrangement (this involves non-Watson Crickbase-pairing). In general, either a run of two or more contiguous Gs ora hexameric region in which >50% of the bases are Gs, is needed for anODN to form a G-tetrad. The longer the run of continuous Gs, and thehigher the G content of the ODN, the higher the likelihood of G-tetradformation, as reflected by higher ellipticity values. Oligonucleotidesthat form G-tetrads can also form higher-level aggregates that are moreeasily recognized and taken up by immune cells, for example, throughscavenger receptors or by nucleolin.

The formation of G-tetrads yields a complex with different physicalproperties than the individual oligonucleotides. Spectroscopically, thisis manifested by an increase in circular dicroism (CD), and an increasein peak absorbance to the 260-280 nm wavelength owing to the formationof secondary structures. Thus, a convenient method for identifyingoligonucleotides that form G-tetrads is to study their CD values. Anincrease in peak ellipticity values to greater than 2.0 is typical of aG-tetrad forming oligonucleotide. For instance, G-tetrad-forming ODNscan have CD values of about 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.5, 4.0, 4.5,5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or higher. The higher theellipticity value, the greater the tetrad-forming capacity of theoligonucleotide, so an ODN with a CD value of about 8.5 is typicallymore suppressive than an ODN with a CD value of about 2.9.

In some embodiments, the ODN is from about 8 to about 100 nucleotides inlength. In particular examples, the ODN is from about 10 to about 30nucleotides in length. Optionally, the suppressive ODN has multipleguanosine-rich sequences, for example, in certain embodiments the ODNhas from about two to about 20 guanosine-rich sequences, or, moreparticularly, from about two to about four guanosine-rich sequences.

In one embodiment, the suppressive ODNs have a sequence comprising atleast one of the human telomere-derived TTAGGG suppressive motifs (seeExample 1). In some examples, the ODN has at least one TTAGGG motif, andin certain examples, the ODN has multiple TTAGGG motifs. For example, inparticular examples, the ODN has from about two to about 20 TTAGGGmotifs, or from about two to about four TTAGGG motifs. In thisembodiment, suppressive ODNs containing multiple TTAGGG repeats are themost suppressive. Single TTAGGG motifs are suppressive only whenincorporated into larger ODNs with greater than 10 bases.

Suppression of CpG-induced immune activation requires a G-tetrad-formingsequence that imposes the two-dimensional structure necessary forG-tetrad formation. Examples of suppressive ODN include, but are notlimited to, those shown in FIG. 9. However, any oligonucleotide capableof forming G-tetrads may be used to suppress CpG DNA-induced immuneactivation. In particular examples, the ODN includes selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, and SEQ ID NO: 25.

Furthermore, in particular embodiments the ODN is modified to preventdegradation and increase stability. In one embodiment, suppressive ODNscan include modified nucleotides to confer resistance to degradation.Thus, because phosphorothioate-modified nucleotides confer resistance toexonuclease digestion, a suppressive ODN can be “stabilized” byincorporating phosphorothioate-modified nucleotides.

In some embodiments, the ODN has a phosphate backbone modification, andin particular examples, the phosphate backbone modification is aphosphorothioate backbone modification. In one embodiment, theguanosine-rich sequence and its immediate flanking regions includephosphodiester rather than phosphorothioate nucleotides. In one specificnon-limiting example, the sequence TTAGGG includes phosphodiester bases.In some examples, all of the bases in an ODN are phosphodiester bases.In other examples, the ODN is a phosphorothioate/phosphodiester chimera.

As disclosed herein, any suitable modification can be used to render theODN resistant to degradation in vivo (for example, via an exo- orendo-nuclease). In one specific, non-limiting example, a modificationthat renders the ODN less susceptible to degradation is the inclusion ofnontraditional bases such as inosine and quesine, as well as acetyl-,thio- and similarly modified forms of adenine, cytidine, guanine,thymine, and uridine. Other modified nucleotides include nonionic DNAanalogs, such as alkyl or aryl phosphonates (for example, those in whichthe charged phosphonate oxygen is replaced with an alkyl or aryl group,as set forth in U.S. Pat. No. 4,469,863), phosphodiesters andalkylphosphotriesters (for example, those in which the charged oxygenmoiety is alkylated, as set forth in U.S. Pat. No. 5,023,243 andEuropean Patent No. 0 092 574). ODNs containing a diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both termini,have also been shown to be more resistant to degradation.

The suppressive ODN of the disclosure can be synthesized by standardmethods well known in the art. Most commonly, synthesis is performed onan oligonucleotide synthesizer using the standard cyanoethylphosphoramidite chemistry. These include, but are not limited to,phosphodiester, phosphorothioate, peptide nucleic acids, syntheticpeptide analogues, and any combination thereof. Those skilled in the artwill recognize that any other standard technique may be used tosynthesize the suppressive ODN described herein.

The suppressive ODN can be included in a single polynucleotide that alsoincludes an immunostimulatory CpG motif, such as a D or a K typeoligonucleotide sequence. Generally, if a suppressive ODN sequence andan immunostimulatory ODN sequence are included in the a singlepolynucleotide, the polynucleotide selectively inhibits or suppressesimmune activation.

In one example, a polynucleotide that selectively inhibits or suppressesimmune activation includes an immunostimulatory CpG motif placed 5′ to asuppressive ODN sequence. In this embodiment, a spacer of at least 10nucleotides is included between the CpG motif and the suppressive ODNsequence in the polynucleotide. The spacer can be any nucleotidesequence of interest, provided it does not include an immunostimulatoryCpG motif or the sequence of a suppressive ODN. In one embodiment, thesingle polynucleotide includes an immunostimulatory CpG motif placed 5′to a suppressive ODN sequence, wherein the immunostimulatory CpG motifis separated from the suppressive ODN sequence by at least 10nucleotides. Thus, in several examples, the immunostimulatory CpG motifis separated from the suppressive ODN sequence by at least about 10,about 15, about 20, about 25, about 50 or about 100 nucleotides in asingle polynucleotide. Spacers of about at least about 100 nucleotides,at least about 500 nucleotides, or at least about a kilobase can also beutilized.

A polynucleotide that selectively inhibits or suppresses immuneactivation does not include an immunostimulatory CpG motif placedimmediately adjacent (5′) to a suppressive ODN sequence. Thus, apolynucleotide that selectively inhibits or suppresses immune activationdoes not include a 5′ immunostimulatory CpG motif separated by less than9 bases from a 3′ suppressive ODN sequence.

In another example, a polynucleotide that selectively inhibits orsuppresses immune activation includes an immunostimulatory CpG motifplaced 3′ to a suppressive ODN sequence. The immunostimulatory CpG motifcan be placed immediately adjacent (3′) to a suppressive ODN. In oneexample, a polynucleotide that selectively inhibits or suppresses immuneactivation can include a 3′ immunostimulatory CpG motif separated byless than 9 bases from a 5′ suppressive ODN. In another example, aspacer of any length can be included between the 3′ immunostimulatoryCpG motif and the 3′ suppressive ODN. In a further example, a spacer ofat least one, at least two, at least ten, at least 100, or severalkilobases can be inserted between the 3′ immunostimulatory CpG motif andthe suppressive ODN. Thus, any oligonucleotide including animmunostimulatory CpG motif positioned 3′ of a suppressive ODN sequenceselectively inhibits or suppresses immune activation. In addition, anydouble stranded nucleic acid that includes a suppressive ODN sequenceand immunostimulatory CpG motif in trans (on opposite strands of thedouble stranded nucleic acid) selectively inhibits or suppresses immuneactivation.

In one embodiment, a suppressive ODN is included in a delivery complex.The delivery complex can include the suppressive ODN and a targetingmeans. Any suitable targeting means can be used. For example, in someembodiments, a suppressive ODN is associated with (for example,ionically or covalently bound to, or encapsulated within) a targetingmeans (for example, a molecule that results in higher affinity bindingto a target cell, such as a B cell). A variety of coupling orcross-linking agents can be used to form the delivery complex, such asprotein A, carbodiamide, and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Examples of oligodeoxynucleotide delivery complexesinclude a suppressive ODN associated with a sterol (for example,cholesterol), a lipid (for example, a cationic lipid, anionic lipid,virosome or liposome), and a target cell specific binding agent (forexample, a ligand recognized by target cell specific receptor). Withoutbeing bound by theory, the complex is sufficiently stable in vivo toprevent significant uncoupling prior to delivery to the target cell. Inone embodiment, the delivery complex is cleavable such that the ODN isreleased in a functional form at the target cells.

B. Pharmaceutical Compositions

The suppressive ODNs described herein may be formulated in a variety ofways depending on the location and type of disease to be treated.Pharmaceutical compositions are thus provided for both local (forexample, intra-articluar) use as well as for systemic use. Therefore,the disclosure includes within its scope pharmaceutical compositionscomprising at least one suppressive ODN formulated for use in human orveterinary medicine.

Pharmaceutical compositions that include at least one suppressive ODN asdescribed herein as an active ingredient, or that includes both asuppressive ODN and additional agents, such as anti-inflammatory oranti-arthritis factors as active ingredients, can be formulated with anappropriate solid or liquid carrier, depending upon the particular modeof administration chosen. Additional active ingredients include, forexample, biological response modifiers, such KINERET® (anakinra),ENBREL® (etanercept), or REMICADE® (infliximab), a disease-modifyingantirheumatic drug (DMARD), such as ARAVA® (leflunomide), a steroid,such as prednisone or cortisone, a nonsteroidal anti-inflammatory drug(NSAID) such as celecoxib, choline magnesium trisalicylate, diclofenac,diclofenac potassium, diclofenac XR, diflunisal, etodolac, etodolac ER,fenoprofen, flurbiprofen oral, ibuprofen, indomethacin, indomethacin SR,indomethacin suppositories, ketoprofen, ketoprofen ER, meclofenamate,meloxicam, nabumetone, naproxen, naproxen CR, naproxen ER, oxaprozin,piroxicam, rofecoxib, salsalate, sulindac, or tolmetin sodium, or otherproducts, such as HYALGAN® (hyaluronan) and SYNVISC® (hylan G-F20).

The pharmaceutically acceptable carriers and excipients useful in thisdisclosure are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable fluid vehicles such as water, physiologicalsaline, other balanced salt solutions, aqueous dextrose, glycerol or thelike. Excipients that can be included are, for instance, proteins, suchas human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered may also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical and oral formulations can be employed.Topical preparations can include eye drops, ointments, sprays and thelike. Oral formulations may be liquid (for example, syrups, solutions,or suspensions), or solid (for example, powders, pills, tablets, orcapsules). For solid compositions, conventional non-toxic solid carrierscan include pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. Actual methods of preparing such dosage forms areknown, or will be apparent, to those of ordinary skill in the art.

The pharmaceutical compositions that comprise a suppressive ODN, in someembodiments, will be formulated in unit dosage form, suitable forindividual administration of precise dosages. The amount of activecompound(s) administered will be dependent on the subject being treated,the severity of the affliction, and the manner of administration, and isbest left to the judgment of the prescribing clinician. Within thesebounds, the formulation to be administered will contain a quantity ofthe active component(s) in amounts effective to achieve the desiredeffect in the subject being treated.

C. Therapeutic Uses

A method is disclosed herein for treating or preventing an inflammatoryarthropathy in a subject. Inflammatory arthropathies include, but arenot limited to reactive arthritis and rhematoid arthritis. The methodincludes administering a therapeutically effective amount of thesuppressive ODN to a subject having or at risk of developinginflammatory arthropathy, thereby treating or preventing theinflammatory arthropathy. In one embodiment, the suppressive ODN can beadministered locally, such as by intra-articular injection. In anotherembodiment, the suppressive ODN is administered systemically.

In order to treat or prevent an inflammatory arthropathy, atherapeutically effective amount of a suppressive ODN (see above) isadministered to the subject. In one embodiment, the ODN has a CD valueof greater than about 2.9, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, or 5.0. In someembodiments of the method, the ODN is from about 8 to about 100nucleotides in length. In particular examples of the method, the ODN isfrom about 10 to about 30 nucleotides in length. Optionally, thesuppressive ODN has multiple guanosine-rich sequences, for example, incertain embodiments of the method, the ODN has from about two to about20 guanosine-rich sequences, or, more particularly, from about two toabout four guanosine-rich sequences.

In some examples of the method, the ODN has at least one TTAGGG motif,and in certain examples, the ODN has multiple TTAGGG motifs. Forexample, in particular examples of the method, the ODN has from abouttwo to about 20 TTAGGG motifs, or from about two to about four TTAGGGmotifs. In particular examples of the method, the ODN has a sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.

Combinations of these suppressive ODN are also of use. Thus, in oneembodiment, more than one suppressive ODN, each with a different nucleicacids sequence, are administered to the subject. In several specific,non-limiting examples, at least two, at least three, or at least foursuppressive ODN are administered to the subject.

In another embodiment an additional anti-inflammatory agent isadministered in conjunction with a suppressive ODN. The agent can be animmunosuppressive, or an anti-arthritis agent. The administration of theanti-arthritis agent and the suppressive ODN can be sequential orsimultaneous.

In particular examples, the anti-arthritis agent is a biologicalresponse modifier, such as KINERET® (anakinra), ENBREL® (etanercept), orREMICADE® (infliximab), a disease-modifying antirheumatic drug (DMARD),such as ARAVA® (leflunomide), a steroid, such as prednisone orcortisone, a nonsteroidal anti-inflammatory drug (NSAID), such ascelecoxib, choline magnesium trisalicylate, diclofenac, diclofenacpotassium, diclofenac XR, diflunisal, etodolac, etodolac ER, fenoprofen,flurbiprofen oral, ibuprofen, indomethacin, indomethacin SR,indomethacin suppositories, ketoprofen, ketoprofen ER, meclofenamate,meloxicam, nabumetone, naproxen, naproxen CR, naproxen ER, oxaprozin,piroxicam, rofecoxib, salsalate, sulindac, or tolmetin sodium, oranother product, such as HYALGAN® (hyaluronan) or SYNVISC® (hylanG-F20).

Thus, the suppressive ODNs disclosed herein may be administered to asubject for the treatment of inflammatory arthropathies in thatindividual. ODN administration can be systemic or local. Localadministration of the ODN is performed by methods well known to thoseskilled in the art. By way of example, one method of administration tothe knee, hip and/or shoulder of an individual is by intra-articularinjection. For administration to the knee, for example, the joint to beinjected is washed with a betadine solution or other antiseptic. Asolution of an anesthetic, such as about one percent lidocainehydrochloride is injected into the skin and subcutaneous tissue. A 3-waystopcock/needle assembly is utilized to administer the compound via an18-30 gauge needle. The ODN is injected into the joint space using astandard lateral approach well known to those skilled in the art. Theneedle and needle tract are cleansed by flushing with 1% lidocainehydrochloride through the 3-way stopcock assembly as the needle iswithdrawn. The knee is then moved through a flexion-extension arc andthen immobilized in full extension. The patient is then confined to bedfor approximately 24 hours to minimize movement and minimize leakage ofODN from the joint.

In other embodiment, the administration of the suppressive ODN issystemic. Oral, intravenous, intra-arterial, subcutaneous,intra-peritoneal, intramuscular, and even rectal administration iscontemplated.

Other embodiments are methods of treating or preventing inflammatoryarthropathies in a subject that involves contacting immune cells with asuppressive ODN, and transferring the immune cells to a subject havingor at risk of developing inflammatory arthropathies, thereby treating orpreventing the inflammatory arthropathies. Without being bound bytheory, these immune cells act to suppress immune activation in asubject. One specific, non-limiting example is dendritic cells. Thus, incertain examples, the immune cells, such as dendritic cells, arecontacted with a suppressive ODN, and subsequently administered to asubject. The immune cells can be delivered alone, in conjunction with asuppressive ODN, and/or in conjunction with an additionalimmunosuppressive agent. The immune cells can be delivered eithersystemically or locally. In several specific, non-limiting examples, thecells are delivered parenterally, intravenously, intramuscularly,sub-cutaneously, or intra-articularly.

Precise, effective quantities of cells can be readily determined bythose who are skilled in the art and will depend, of course, upon theexact condition being treated by the particular therapy being employed.The cells can be transplanted to a desired location, or can beadministered intravenously. Other agents, such as growth factors orimmunosuppressive agents, can be administered in conjunction with theimmune cells.

D. Kits

Further embodiments of the disclosure include kits useful foradministering the suppressive ODN described herein in vivo or in vitro.For example, a kit useful for treating a subject with inflammatoryarthropathies would comprise an appropriate dosage of suppressive ODN,as well as, optionally, any agents useful for enhancing the inhibitoryeffect of suppressive ODN. Other embodiments further includeinstructions for using the kit, and/or pre-filled syringes foradministering the ODN to a subject.

Thus, in one embodiment, a kit is provided including a container ofsuppressive ODN (a sufficient amount for either a single use or multipleuses), and instructions the suppressive ODN. The instructions can be inwritten form, or can be provided in an electronic format, such as on adiskette or a CD-ROM. Instructions can also be provided in the form of avideo cassette.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

EXAMPLES Example 1 Reduction of CpG-Induced Arthritis by SuppressiveOligodeoxynucleotides

This example demonstrates the influence of CpG DNA and suppressive ODNson the propensity of the host to develop arthritis, and indicates thatthe mechanism of suppressive ODN action is mediated by Cd11c positivecells.

A. General methods

Animals:

Female BALB/c mice were obtained from the Jackson Laboratories (BarHarbor, Me.). The studies were conducted using 8-20 week old mice.

Oligodeoxynucleotides:

ODNs were synthetically produced. Sequences of the phosphorothioate ODNused were:

CpG: GCTAGACGTTAGCGT (SEQ ID NO: 30) suppressive: CCTCAAGCTTGAGGGG (SEQID NO: 1) control: GCTAGATGTTAGCGT. (SEQ ID NO: 31)All ODN were free of detectable protein or endotoxin contamination.Experimental Protocol:

25 μg of ODN in 6 μl of PBS was injected into the knee joint using a 30gauge needle. In some studies, knees were re-injected with PBS or 25 μgof suppressive ODN 24-48 hours after initial CpG ODN administration.Joint swelling was measured in the coronal plane using a micrometercaliper. Histologic analysis was performed by a blinded investigator onfixed, decalcified and paraffin embedded sections stained withhematoxylin/eosin.

TNFα Assays:

Single spleen cell suspensions were prepared in RPMI-1640 supplementedwith 5% heat inactivated fetal calf serum, 1.5 mM L-glutamine and 100U/ml of penicillin/streptomycin. 5×10⁵ cells/well were cultured inflat-bottomed microtiter plates (Costar, Corning, N.Y.) with ODN for 72hr. TNFα levels in culture supernatants were measured by ELISA. Inbrief, 96-well Immulon H2B plates were coated with anti-TNFα Ab(Genzyme, Cambridge, Mass.). Plates were blocked with PBS-1% BSA andoverlaid with culture supernatants. Bound cytokine was detected by theaddition of biotin labeled anti-TNFα Ab (Genzyme, Cambridge, Mass.)followed by phosphatase-conjugate avidin.

RT-PCR:

Total RNA was extracted from homogenized knees using TRIzol reagent(GibCO Life Technologies, Gaithersburg, Md.) as recommended by themanufacturer. 5 μg of total RNA was reverse transcribed into cDNA, whichwas assayed for TNFα (sense ATGAGCACAGAAAGCATGATC; SEQ ID NO:32,antisense TACAGGCTTGTCACTCGAATT; SEQ ID NO:33, 275 bp) and β-actin(sense GACATGGAGGAGTCTGGCACCACA; SEQ ID NO:34, antisenseATCTCCTGCTCGAAGTCTAGAGCAA; SEQ ID NO:35, 440 bp) by PCR as previouslydescribed (Takeshita et al., Neuroreport (2001) 12:3029-3032). Relativeband intensity was determined by ethidium bromide staining of 1% agarosegels using NIH-Image software.

Statistical Analysis:

Statistically significant differences between two groups were determinedusing the Wilcoxon Rank Sum Test. When comparing more than two groups, atwo-tailed non-parametric ANOVA with Dunn's post-test analysis was used.Differences in joint diameters were analyzed by repeated-measures ANOVAusing the Proc Mixed procedure from the Statistical Analysis System(SAS). A p value of <0.05 was considered significant.

B. Induction of Arthritis by CpG ODN

Microbial infection of the gastrointestinal (GI) or genitourinary (GU)tracts is associated with the development of reactive arthritis inhumans. Evidence indicates that bacterial DNA contributes to thisprocess, since 1) bacterial DNA can be detected in arthritic joints(Braun et al., J. Rheumatol. (2000) 27:2185-2192), and 2) bacterial DNAinduces joint inflammation when injected into the knees of normal mice(Deng et al., Nat. Med. (1999) 5:702-705). Deng et al. established thatimmunostimulatory CpG motifs were the cause of this inflammation byshowing that CpG-containing oligodeoxynucleotides (ODN) induced diseasein a manner similar to that induced by purified bacterial DNA (Deng etal., supra). This effect is consistent with the proinflammatoryproperties of CpG ODN, including their ability to stimulate immune cellsto proliferate, differentiate, and secrete proinflammatory chemokinesand cytokines (Deng et al., supra; Klinman et al., Springer Semin.Immunopathol. (2000) 22:173-83).

Consistent with the findings of Deng et al. (Deng et al., ArthritisRheum. (2000) 43:356-364), BALB/c knees injected with CpG ODN developedinflammatory arthritis within 24 hr that peaked after 3-7 days (FIG. 2).CpG-induced arthritis was characterized by swelling (0.14+0.04milimeters (mm) versus 0.02+0.02, p=0.054) and histological changes thatincluded perivascular infiltration by mononuclear cells and hyperplasiaof the synovial lining (FIG. 2). These inflammatory effects wereCpG-specific and localized, since no disease was observed incontra-lateral knees injected with PBS or control ODN. Similar swellingand histologic changes were observed in knees injected with bacterialDNA.

C. Suppressive ODN Block the Development of CpG-Induced Arthritis

To determine whether suppressive ODN prevent CpG mediated inflammatoryarthritis, knees were co-injected with 25 μg of suppressive plus 25 μgof CpG ODN. The inclusion of suppressive ODN reduced swelling from0.14+0.04 mm to 0.02+0.02 mm (p=0.004) and the inflammatory score from1.94+0.32 to 0.67+0.12 (p=0.018, FIG. 2). When joint inflammation wasassessed by magnetic resonance imaging, suppressive ODN reduced CpG ODNinduced fluid accumulation from 95.4+8.2 MR-signal intensity units to52.3+6.7 units (n=5, p<0.001). These effects were specific, sinceco-administering PBS or control ODN had no impact on CpG-inducedarthritis (FIG. 2). In parallel studies, suppressive ODN prevented thearthritis induced by bacterial DNA but not lipopolysaccharide (LPS).

D. Kinetics of the ODN Anti-Inflammatory Effect

To examine the kinetics of this anti-inflammatory effect, suppressiveODN were administered 0, 24 and 48 hours after CpG ODN injection. Tocontrol for the effect of multiple injections, the contralateral kneewas injected with PBS, and the difference in swelling between the twojoints evaluated daily. A significant reduction in swelling was observedwhen joints were treated with suppressive ODN up to two days after CpGadministration (p=0.012, FIG. 2C). However, maximal control of arthritisrequired early intervention (p=0.011, D0 vs D2).

E. Suppressive ODNs Reduce Intra-Articular TNF Production

Previous studies established that the magnitude of CpG-induced arthritiscorrelated with intra-articular TNFα levels (Deng et al., Arthritis Res.(2001) 3:48-53). Consistent with TNFα playing a critical role in thedisease process, TNFα knock out mice fail to develop CpG-inducedarthritis (Deng et al., Arthritis Rheum. (2000) 43:356-364; Ronaghy etal., J. Immunology (2002) 168:51-56). To evaluate whether suppressiveODN had an effect on TNFα production, BALB/c spleen cells werestimulated in vitro with CpG+suppressive ODN. As seen in FIG. 3,suppressive ODN reduced TNFα production in a dose-dependent manner,whereas control ODN had no effect.

To monitor the in vivo effect of suppressive ODN on TNF production,cytokine mRNA levels were measured in the joint. Consistent with earlierreports (Deng et al., Arthritis Rheum. (2000) 43:356-364; Ronaghy etal., J. Immunology (2002) 168:51-56; Kyburz et al., Arthritis Rheum.(2001) 44 (Suppl): S396), CpG ODN up-regulated local TNF mRNA levels(FIG. 3C, D). Co-administering suppressive ODN reduced TNF mRNA by >50%(p<0.003, FIG. 3).

Example 2 Systemic Effect of Stimulatory and SuppressiveOligodeoxynucleotides on the Induction of Inflammatory Arthritis

Animals:

Female BALB/c mice were obtained from the Jackson Laboratories (BarHarbor, Me.). The mice were used at 8-20 weeks of age and were housedunder specific pathogen free conditions.

Oligonucleotides:

ODN used in this study had a phosphorothioate backbone, and werechemically synthesized. They contained <0.1 endotoxin units (EU) ofendotoxin per mg of ODN, as assessed by a Limulus amebocyte lysate assay(QCL-1000, BioWhittaker). The sequence of the CpG ODN wasGCTAGACGTTAGCGT (SEQ. ID NO: 30), of the suppressive ODN:CCTCAAGCTTGAGGGG (SEQ. ID NO: 1), and of the control ODN:GCTAGATGTTAGCGT (SEQ ID NO: 31).

Experimental Protocols:

Arthritis was induced as previously described in Zeuner et al.,Arthritis Rheum. (2002). Briefly 1 or 25 μg of ODN in 6 μl of PBS wasinjected into the knee joint using a 30 gauge needle. Joint swelling wasmeasured in the coronal plane using a micrometer caliper (General ToolsMfg CO, NY, N.Y.). Mice were euthanized on day 4 and the knees fixed,decalcified, sectioned, and stained with hematoxylin/eosin prior tohistologic examination. Scoring was performed by a blinded investigatorusing a scale of 0-4. 0=absence of inflammation, 1=sparse, localizedperivascular infiltrate, 2=moderate infiltrate, 3=moderate-denseinfiltrate with synovial hyperplasia, 4=dense infiltrate with pronouncedsynovial hyperplasia.

In some experiments, donor mice were injected intraperitoneally (i.p.)with 300 μg of ODN in PBS. A single cell suspension prepared from thespleens of these mice was prepared, and 20×10⁶ transferred intravenously(IV) to naive littermate recipients. These spleen cells were enriched ordepleted of various subpopulations using magnetic bead separation(Vario-Macs System, Miltenyi) as recommended by the manufacturer.

Statistical Analysis:

Differences between two groups determined using the Wilcoxon Rank SumTest. Differences between multiple groups were evaluated using atwo-tailed analysis of variance (ANOVA) on Ranks with Dunn's post testanalysis. Differences in joint diameters were compared byrepeated-measures ANOVA. A p value of <0.05 was considered significant.Data are presented as mean+/−SEM.

B. Effect of Systemic CpG ODN on Sensitivity of Joints to InflammatoryStimuli

Previous studies established that intra-articular injection of bacterialDNA or CpG ODN induces arthritis in mice, characterized by aninflammatory cell infiltrate, perivascular accumulation of mononuclearcells, and hyperplasia of the synovial lining (Deng and Tarkowski,Arthritis Rheum. (2000) 43:356-364). Since reactive arthritis in humansis associated with infection of the gastrointestinal (GI) orgenitourinary (GU) tract rather than the joint, we explored whether CpGDNA in the systemic circulation might alter the sensitivity of joints toinflammatory stimuli.

To examine this possibility, normal BALB/c mice were injected IP with300 μg of CpG DNA, and then challenged with a sub-arthritogenic dose oflocal CpG ODN. As seen in FIG. 5, normal mice and mice treated with PBSor control ODN did not develop arthritis (no inflammation, no swelling)when injected with 1 μg of CpG. In contrast, animals pre-treated withsystemic CpG DNA developed significant joint swelling, a mononuclearcell infiltrate, and synovial hyperplasia when challenged with the samedose of CpG ODN. These changes were triggered by exposure to local CpGDNA, as no inflammation developed when the joints of systemicallytreated animals were injected with PBS.

These findings indicate that CpG DNA in the systemic circulation(perhaps released by dying bacteria in the GI or GU tract) can increasethe host's susceptibility to the development local arthritis.

C. Systemic Administration of Suppressive ODN Reduces Susceptibility toArthritis

It has been demonstrated that suppressive ODN (containing motifs thatselectively inhibit the immunostimulatory activity of CpG ODN)significantly reduce the swelling, synovial hyperplasia and leukocyteinfiltration induced by CpG DNA. These effects were observed whensuppressive ODN were injected directly into arthritic joints. Becausesystemic CpG DNA can increase the host's susceptibility to arthritis,suppressive ODN in the systemic circulation might lower thissusceptibility.

To test this hypothesis, BALB/c mice were treated intraperitoneally with300 μg of suppressive ODN 0-3 days prior to the intra-articularinjection of 25 μg CpG DNA. Consistent with previous studies, naive mice(and mice pre-treated with control ODN or PBS) developed severearthritis following local CpG ODN challenge (FIG. 6). In contrast,systemic administration of suppressive ODN three days prior to local CpGDNA challenge reduced joint swelling and inflammation by 80-85% (FIG. 7;p<0.029). Unlike the situation with locally administered suppressiveODN, which reduced inflammation when administered up to two days afterthe induction of arthritis, suppressive ODN were effective systemicallyonly if delivered several days prior to the induction of arthritis.These findings demonstrate that instead of blocking CpG inducedarthritis locally, suppressive ODN in the systemic circulation activatea regulatory cascade that requires several days to mature.

D. Spleen Cells from Suppressive ODN Treated Mice Prevent CpG-InducedArthritis

Spleen cells from mice treated systemically with suppressive ODN weretransferred to naive controls, which were then injected with 25 μg ofCpG DNA intra-articularly. As expected, spleen cells from untreateddonors had no effect on the development of CpG-induced arthritis (FIG.8). By comparison, the transfer of 20×10⁶ spleen cells from suppressiveODN treated donors significantly reduced joint swelling and inflammationin the recipients. These findings indicate that systemicallyadministered suppressive ODN elicit a population of regulatory cellsthat inhibit CpG-induced arthritis.

To define the time course over which these regulatory cells aregenerated, splenocytes were isolated from one to three days aftertreatment with suppressive ODN. Cells from animals treated withsuppressive ODN for three days significantly inhibited CpG-inducedarthritis, whereas splenocytes from animals exposed to suppressive ODNfor shorter periods were progressively less effective.

To characterize the cell type responsible for this resistance toCpG-induced arthritis, magnetic beads were used to deplete or enrichspecific cell subpopulations from donor spleens. Depletion of CD19⁺ Bcells, T cells, or NK cells had no effect on CpG-induced arthritis (FIG.9). However, removal of CD11c⁺ dendritic cells resulted in a completeabrogation of the suppressive activity of the transferred spleen cellpopulation. Similarly, the transfer of only 5×10⁵ CD11c⁺ enriched cellsfrom suppressive ODN treated mice to normal recipients conferredresistance to CpG-induced arthritis.

Example 3 Repetitive Elements Present in Mammalian Telomeres SuppressBacterial DNA-Induced Immune Activation

This example demonstrates the ability of TTAGGG multimers to inhibitCpG-induced immune activation.

A. General Methods

Reagents

Endotoxin-free phosphorothioate or phosphodiester ODNs were chemicallysynthesized. 7-DG modified ODNs were synthesized using the10-camphorsulphonyl-oxaziridine oxidization protocol recommended by themanufacturer (Glen Research, Sterling, Va.). Mammalian DNA was isolatedfrom calf thymus and murine spleen (WIZARD® Genomic DNA purificationkit, Promega Corporation, Madison, Wis.). E. coli was obtained fromSIGMA-ALDRICH® (St. Louis, Mo.). Telomerase knockout mice were obtainedfrom Johns Hopkins Univ., Baltimore, Md. All DNA obtained fromcommercial providers was re-purified to eliminate endotoxin (<0.1 U/mg).Double stranded DNA was converted to single stranded DNA by heatdenaturing at 95° C. for five minutes followed by cooling on ice. BAL-31(NEW ENGLAND BIOLABS®, Beverly, Mass.) digestion of CT DNA was continuedfor 2 hours at 30° C. according to manufacturer's recommendations. Atthe end of the incubation, the enzyme was inactivated at 65° C. for 10minutes. Plasmid encoding for 1.6 kb long TTAGGG repeat was obtainedfrom University of Texas Southwestern Medical Center, Dallas, Tex.Non-telomere coding plasmid was from Vical (San Diego, Calif.).

Mice

Specific pathogen-free male BALB/c mice (Jackson Laboratories, BarHarbor, Me.) were housed in sterile micro-isolator cages in a barrierenvironment and injected intraperitoneally with 400 μg of CpG ODN plus200 μg of suppressive or control ODN. Spleen cells were harvested 6hours later and monitored for cytokine production after 36 hours. Inorder to measure the pEPO transgene expression levels, female Balb/cmice (4-6 weeks old) were injected with 30 μg of pVRmEPO (Vical, SanDiego, Calif.) plasmid in sterile saline into the anterior tibialismuscle alone or in combination with 50 μg of control or suppressive ODN.Hematocrits were measured as described (Tripathy et al., Proc Natl AcadSci USA. 93:10876-80, 1996) on blood collected by tail vein puncture.

Cytokine and IgM ELISA Assays

Immulon 2 microtiter plates (Dynex Technologies Inc., Chantilly Va.)were coated with anti-cytokine or anti-IgM antibodies (Pharmingen, SanDiego Calif.) and then blocked with PBS-1% bovine serum albumin (BSA).Serially diluted culture supernatant or serum was added for two hours.Cytokine was detected using biotinylated anti-cytokine antibody followedby phosphatase-streptavidin (Pharmingen) whereas bound IgM was detectedusing phosphatase-conjugated anti-IgM antibodies (Southern BiotechnologyAssociates, Birmingham, Ala.) as described.

Detection of Co-Stimulatory Molecule Expression by FACS

2×10⁶ spleen cells/ml were incubated with ODN for 24 hours. Cells werewashed, fixed with 5% paraformaldehyde for 15 minutes, and stained withPE-labeled anti CD-40, anti CD-86, and anti ICAM-1 (Pharmingen, SanDiego, Calif.) for 30 minutes at room temperature. Cells were washed,re-suspended in PBS/BSA (supplemented with Azide), and analyzed byfluorescence activated cell sorting (FACSort, Becton Dickinson, SanJose, Calif.).

Cytokine RT-PCR

Spleen cells were isolated 6 hours after CpG ODN injection. Total RNAwas extracted, reverse-transcribed, and amplified in a standard PCRreaction for 24 cycles using primers specific for murine interleukin-6(IL-6), interleukin-12 (IL-12), and interferon (IFN) as previouslydescribed. PCR amplified material was separated on 1.5% agarose gels andvisualized under ultraviolet (UV) light after ethidium bromide staining.

In Vitro DNA-PK Assay

DNA-dependent protein kinase activity was measured as recommended by themanufacturer (Promega, Madison, Wis.). Briefly, 20 units ofaffinity-purified human DNA-PKcs and 10 nM of ODN were incubated with0.4 mM biotinylated peptide substrate and P₃₂-ATP for 5 minutes at 30°C. in reaction buffer. Phosphorylated substrate was captured onstreptavidin coated SAM membranes (Promega), and measured in ascintillation counter.

Cell Transfection and Luciferase Assay

HEK 293 (5×10⁴) cells (ATCC, Manassas, Va.) were transfected with 0.8 μgof vector plasmid (pCIneo, Promega, Madison, Wis.), pCIneo-mTLR9, plus0.1 μg of p5xNF-kB-luc (Stratagene, Lajolla, Calif.) and 0.1 μg ofpSV-beta-galactosidase (Promega) and incubated overnight at 37° C. Thecells were then stimulated with indicated ODN for 24 h. Cells were thenharvested and luciferase assay was performed as recommended by themanufacturer (Promega). Beta-galactosidase activity was used tonormalize the data.

Measurement of Circular Dichroism

A Jasco J-720A spectropolarimeter was used to measure the circulardichroism of ODN (50 g/ml in 0.1×PBS). Data is expressed as the meanpeak ellipticity (mdeg/abs) of 5-10 readings/sample in the 260-270 nmrange.

Statistical Analysis

In vitro assays were performed in triplicate on at least three differentspleen cell preparations. All in vivo experiments were performed on aminimum of five to ten mice/group. Statistical significance wasevaluated using Student's t test. Correlation analysis is computed bylinear correlation analysis between CD data vs % suppression.

B. TTAGGG Multimers Inhibit CpG-Induced Immune Activation

The ability of TTAGGG multimers to inhibit CpG-induced immune activationwas tested. Initial experiments showed that ODNs containing suppressivemotifs inhibit CpG-induced immune activation in a dose-dependent fashion(FIG. 10A).

ODNs containing the largest number of TTAGGG repeats (n=4) were the mostsuppressive (p<0.001; FIG. 10B). Single TTAGGG hexamers were suppressiveonly when incorporated into larger ODNs (≧10 bases in length), and weresomewhat less active than TTAGGG multimers. TTAGGG motifs weresuppressive both in cis and in trans; they inhibited immune activationwhen present on the same or on a different strand of DNA than thatexpressing the stimulatory CpG sequence (FIGS. 10C and D). Thisinhibitory activity was exquisitely specific: even high concentrationsof suppressive ODNs had no effect on mitogen-induced immune responses(FIG. 10D), indicating that suppressive ODN were neither toxic nornon-specifically immunosuppressive.

C. Suppression is Mediated by Poly-G Sequences

The bases contributing to this suppressive activity were identified bysystematically modifying ODNs containing single TTAGGG motifs.Substitutions outside the telomere-derived sequence did notsignificantly affect suppression. Replacing multiple bases in the TTAregion of the TTAGGG motif also had little effect on the suppressiveactivity (FIG. 10E). In contrast, replacing two or more of the Gs inthis motif substantially reduced the ODN's ability to block CpG-inducedimmune activation (FIG. 10E). These results indicate that suppression ismediated by the poly-G sequence itself, or the two-dimensional structureimposed by that sequence.

D. Suppression is Mediated by the Two-Dimensional Structure of theMotifs

To differentiate between these alternatives, individual Gs were replacedby 7-deaza guanosine (7-DG) analogues. These 7-DG substitutions did notalter the base sequence of the ODN but did prevent Hoogsteen hydrogenbonding between guanosines, thereby reducing G tetrad formation (Hurleyet al., Trends Pharmacol. Sci. 21:136-142, 2000; Lilley et al., EMBO J.13:993-1001, 1994). The resultant loss in secondary structure isreflected by a loss in circular dichroism (Lilley et al., EMBO J.13:993-1001, 1994). ODNs capable of forming G tetrads typically havepeak CD values >2, while those without such secondary structure havecircular dichroism values <1.4 (FIG. 10).

Introducing a 7-DG substitution significantly reduced an ODN's abilityto form a G-tetrad and to mediate suppression (p<0.001, FIG. 11). Toconfirm that G-tetrad formation was critical to suppression, ODNs weresynthesized that lacked the TTAGGG motif but still formed G-tetrads.These novel ODNs suppressed CpG ODN and bacterial DNA induced immunestimulation by >90% (p<0.001; FIG. 10F). Indeed, there was a consistentcorrelation between G-tetrad forming ability and suppressive activity(R=0.832, FIG. 11B).

E. Mechanism of Suppressive ODN Activity

The mechanism by which suppressive ODNs inhibit immune activation wasexplored. Both bacterial DNA and CpG ODN stimulated DNA-dependentprotein kinase activity in vitro. Of particular interest, this activitywas specifically blocked by TTAGGG multimers and other G tetrad formingODN, but not their 7-DG modified analogues. These findings indicate thatsuppressive motifs inhibit CpG-dependent DNA-PK_(os) activation ofimmune cells. In contrast, suppressive ODNs did not block the binding orinternalization of CpG ODNs mediated by Toll-like receptor 9.

Example 4 Effect of Suppressive ODNs on CpG-Induced Immune Action

This example demonstrates the kinetics, magnitude, and nature of theimmune inhibition elicited by suppressive motifs. Previous studiesestablished that the immunostimulatory activity of CpG DNA can bereversed several hours later either by removing the stimulatory DNA oradding suppressive DNA. The same cells that interact with stimulatorymotifs also recognize suppressive motifs. When both sequence types arepresent on the same strand of DNA, recognition proceeds in a 5′→3′direction. Suppression is generally dominant over stimulation, althougha motif in the 5′ position can interfere with recognition of a motifimmediately downstream. Understanding the rules governing cellularresponses to stimulatory and suppressive motifs facilitates the designof ODN for therapeutic uses.

A. General Methods

Animals:

Female Balb/c mice were obtained from the Jackson Laboratories (BarHarbor, Me.). The mice were housed under specific pathogen freeconditions, and used at 8-20 weeks of age. All studies involvedprotocols approved by the CBER Animal Care and Use Committee.

Oligodeoxynucleotides:

Studies utilized phosphorothioate modified ODNs that were synthesized atthe CBER core facility Verthelyi, D., et al., J Immunol (2001) 166:2372.The following ODNs were used: immunostimulatory ODN1466 (TCAACGTTGA;SEQ. ID NO: 26) and ODN1555 (GCTAGACGTTAGCGT; SEQ. ID NO: 27), controlODN1471 (TCAAGCTTGA; SEQ. ID NO: 28) and ODN1612 (GCTAGAGCTTAGGCT; SEQ.ID NO: 29), suppressive ODN1502 (GAGCAAGCTGGACCTTCCAT; SEQ. ID NO: 20)and ODNH154 (CCTCAAGCTTGAGGGG; SEQ. ID NO: 1). Underlined basesrepresent the 10-mer sequences that were incorporated into complexmulti-determinant ODN used in some experiments. There was no detectableprotein or endotoxin contamination of these ODN.

Mammalian DNA was purified from BALB/c spleens (Wizard Genomic DNApurification kit, Promega, Madison, Wis.). E. coli DNA was obtained fromGibco BRL (Rockville, Md., USA). Endotoxin contamination in thesepreparations was <0.1 U/ml after purification Klinman, D. M., et al., J.Immunol. (1997) 158:3635. Double stranded DNA (dsDNA) was converted tosingle stranded DNA (ssDNA) by heat denaturing at 95° C. for 5′ followedby immediate cooling on ice.

Cytokine ELISA Assays:

Single spleen cell suspensions were washed three times and re-suspendedin RPMI-1640 supplemented with 5% heat inactivated fetal calf serum(FCS), 1.5 mM L-glutamine and 100 U/ml of penicillin/streptomycin. 5×10⁵cells/well were cultured in flat-bottomed microtiter plates (Costar,Corning, N.Y.) with 1 μM ODN for 18-24 h. Culture supernatants werecollected, and cytokine levels measured by ELISA. In brief, 96 wellImmulon H2B plates were coated with cytokine-specific antibodies andblocked with PBS 1% BSA as previously described in Klinman and Nutman,Current Protocols in Immunology (1994), Coligan, Kruisbeek, Margulies,Shevach, and Strober, eds., Greene Publishing Associates, Brooklyn, N.Y.Culture supernatants were added, and bound cytokine detected by theaddition of biotin labeled secondary antibodies followed byphosphatase-conjugated avidin and a phosphatase-specific colorimetricsubstrate (PNPP, Pierce, Rockford, Ill.). Standard curves were generatedusing recombinant cytokines. The detection limit for these assays was:0.8 U/ml for IFNγ, 0.1 ng/ml for IL-6 and 0.1 ng/ml for IL-12. Allassays were performed in triplicate.

Cytokine-Specific ELIspot Assays:

A single spleen cell suspension prepared in RPMI 1640 plus 5% FCS wasserially diluted onto plates pre-coated with anti-cytokine antibodies.(see Klinman and Nutman, Current Protocols in Immunology (1994),Coligan, Kruisbeek, Margulies, Shevach, and Strober, eds., GreenePublishing Associates, Brooklyn, N.Y.). Cells were incubated with 1 μMODN at 37° C. for 8-12 hours, and their secretion of cytokine detectedcolorimetrically as previously described (Klinman and Nutman, CurrentProtocols in Immunology (1994), Coligan, Kruisbeek, Margulies, Shevach,and Strober, eds., Greene Publishing Associates, Brooklyn, N.Y.).

Cell-Surface Binding and Internalization of ODN:

Spleen cells (2×10⁶/ml) were incubated with 1 μM of unlabeled and/orfluorescent-labeled ODN for 10 minutes at 4° C. (binding experiments) orat 37° C. for 1 hour (uptake experiments Gursel et al., J. Leuko. Biol.(2001). Cells were washed, fixed, and analyzed by FACScan (BectonDickinson, San Jose, Calif.).

Statistical Analysis:

Statistically significant differences between two groups were determinedusing the Wilcoxon Rank Sum Test. When comparing more than two groups,differences were determined using a 2-tailed non-parametric ANOVA withDunn's post-test analysis. A p value of <0.05 was consideredsignificant.

B. Mammalian DNA Suppresses CpG DNA-Induced Immune Activation

Single stranded bacterial DNA and synthetic ODN containing unmethylatedCpG motifs stimulate immune cells to mature, proliferate and producecytokines, chemokines, and immunoglobulins. (Klinman et al., Proc. Natl.Acad. Sci. USA (1996) 93:2879; Roman et al., Nature Medicine (1997)3:849; Roman et al., (1997) Nature Medicine 3:849; Yamamoto et al.,(1992) J. Immunol. 148:4072; Krieg et al., (1995) Nature 374:546). Theseeffects can be blocked by “poly-G” and/or “GC” rich DNA motifs (Krieg etal., (1998) Proc. Natl. Acad. Sci. 95:12631; Pisetsky et al., (1995) NYAcad. Sci. 772:152). Scores of ODNs were synthesized and tested, andeventually several were identified that selectively inhibitedCpG-induced immune responses. The two most active of these suppressiveODN (ODN1502 and ODNH154) were selected for detailed study. As seen inFIG. 12, suppressive ODN blocked a majority of the IFNγ productioninduced by bacterial DNA or CpG ODN (p<0.01). Suppressive ODN wereneither toxic nor broadly immunosuppressive, as they did not interferewith the mitogenic activity of LPS or Concavalin A (Con A; FIG. 12).

The activity of suppressive ODNs was concentration dependent, with 50%suppression being achieved at a suppressive to CpG ODN ratio ofapproximately 1:3 (FIG. 13). To examine the kinetics of this inhibition,suppressive ODN were added to BALB/c spleen cells at various times afterCpG-induced stimulation. Maximal inhibition was observed whensuppressive ODN were co-administered with CpG ODN, althoughstatistically significant inhibition persisted when suppressive ODN wereadded up to 3 hours later (FIG. 14). These findings indicate that CpGinduced immune activation is an ongoing process, and can be inhibitedafter the stimulatory signal is delivered.

To test this conclusion, spleen cells were incubated with CpG ODN forvarious periods and cytokine production analyzed after 24 hours. Cellsstimulated with CpG DNA for 8 hours produced 90% as much cytokine ascells stimulated continuously for 24 hours (FIG. 15). Cells treated withCpG ODN for only 4 hours produced half as much cytokine, while cellstreated with CpG DNA for <2 hours showed only minimal activation (FIG.15). These findings support the conclusion that CpG-induced cellularactivation is reversible for several hours.

C. Suppressive ODNs do not Block CpG ODN Uptake or Induce the Productionof Inhibitory Factors

The results described above indicate that CpG-induced immune activationcan be reversed either by adding suppressive ODNs or by removingstimulatory ODNs. This indicates that suppressive ODNs block the ongoinguptake of CpG DNA. Yet FACS analysis demonstrated that neither cellsurface binding nor internalization of fluorescein isothiocyanate(FITC)-labeled CpG ODN was significantly reduced by suppressive ODN atconcentrations that blocked cytokine production by approximately 75%(FIG. 16).

The possibility that suppressive motifs might induce the production of afactor that blocks CpG-dependent immune activation was theninvestigated. Initial studies established that BALB/c spleen cellspre-incubated with suppressive ODN remained unresponsive to CpG-inducedstimulation for several hours (Table I, line 3). If this non-responsivestate was mediated by a soluble factor (or inhibitory cell-cellinteractions) then cells pre-treated with suppressive ODN should blockCpG-induced stimulation of naive spleen. As seen in Table I, cellstreated with suppressive ODN had no significant effect on CpG dependentcytokine production by fresh splenocytes.

TABLE I Effect of mixing cells treated with suppressive versusstimulatory ODN Cells pre- ODN added % maximal treated with Fresh duringcytokine production suppressive ODN cells culture IL-6 IL-12 − + CpG 100± 13  100 ± 6  − + Control 3 ± 2 7 ± 2 + − CpG 9 ± 6 6 ± 2 + − Control 0± 0 0 ± 0 + + CpG 86 ± 16 105 ± 12  + + Control 0 ± 0 0 ± 0 BALB/cspleen cells were treated with 1 μM suppressive ODN₁₅₀₂ for 2 hours andthen washed (first column). These cells were added to naive splenocytes(second column) plus 1 μM of control ODN₁₄₇₁ or CpG ODN₁₅₅₅. IL-6 andIL-12 levels in culture supernatants were measured by ELISA after 18hours. Results represent the average + SD of triplicate assays, eachstandardized to the response induced by bacterial DNA (62 pg/ml IL-6;134 pg/ml IL-12).D. Cellular Recognition of Suppressive Versus Stimulatory Motifs

The above studies establish that suppressive motifs on one strand of DNAblock the immune activation induced by stimulatory motifs on a differentstrand (trans suppression). To better understand the interaction betweensuppressive and stimulatory motifs, ODNs containing both weresynthesized. In the simplest case, a 20-mer was constructed in which aCpG motif was placed immediately 5′ to a suppressive motif (referred toas [CpG−Sup] ODN).

Experiments showed that this ODN was stimulatory, triggering murinespleen cells to produce IL-6, IL-12 and IFNγ to the same extent as anODN of the same length in which the suppressive motif was replaced by a‘control’ sequence (one that was neither stimulatory nor suppressive,Table II). [CpG−Sup] ODNs also failed to block the immune activationinduced by an independent CpG ODN (Table II). These results indicatethat a suppressive motif is inactive when located immediately 3′ to aCpG motif on the same strand of DNA. Similar results were obtained instudies of additional [CpG−Sup] ODNs that utilized different stimulatoryand suppressive motifs.

TABLE II Effect of motif position on immunostimulatory activity Locationof motifs # of cytokine secreting cells (5′ → 3′) IL-6 IL-12 IFNg CpGODN* 79 ± 13 1980 ± 230 260 ± 40 [CpG - Sup] ODN* 72 ± 14 2080 ± 480 230± 60 [Sup-CpG] ODN 0 ± 0 140 ± 30  0 ± 0 [CpG-Cont] ODN* 64 ± 12 2210 ±130 284 ± 34 [Cont-CpG] ODN* 80 ± 11 1942 ± 88  238 ± 28 [Cont-Sup] ODN8 ± 2 184 ± 34 36 ± 8 [CpG-Sup] ODN + Sup 4 ± 2 226 ± 38 28 ± 6 ODN[Sup-CpG] ODN + CpG 7 ± 3 250 ± 32 34 ± 9 ODN* 10⁶ BALB/c spleen cellswere co-incubated with 1 μM of each ODN. Complex ODN (20 bp in length)were constructed from 10-mers containing suppressive (Sup), stimulatory(CpG) or control (Cont) motifs. The number of cytokine secretingcells/10⁶ was determined by ELIspot after 24 hours of stimulation.Equivalent results were derived using combinations of motifs derivedfrom two different stimulatory (ODN₁₅₅₅ and ODN₁₄₆₆), control (ODN₁₄₇₁and ODN₁₆₁₂) and suppressive (ODN₁₅₀₂ and ODN_(H154)) ODN. Resultsrepresent the average ± SD of triplicate assays involving at least twoODN of each type. *Stimulatory ODN, p < .05.

To better understand this phenomenon, longer ODNs were synthesized inwhich the CpG and suppressive motifs were separated by progressivelylonger CT spacers. Adding a 5 base spacer generated an ODN that wasstill stimulatory (Table III). However, separating the motifs by >10bases yielded ODNs that were suppressive, since they blocked thestimulatory activity of co-administered CpG ODNs (Table III).

TABLE III Effect of distance between motifs on ODN activity Cytokineproducing cells (% maximum) ODN IL-6 IL-12 IFNg CpG ODN * 100 ± 11 100 ±7  100 ± 10  CpG ODN * + Cont ODN  97 ± 14 98 ± 9 100 ± 17  CpG ODN * +Sup ODN 16 ± 6 21 ± 6 18 ± 5  [CpG - Sup] ODN *  87 ± 12 >100 ± 14  92 ±14 [CpG - 5 bases - Sup] ODN * >100 ± 4  >100 ± 21  >100 ± 22  [CpG - 10bases - Sup] ODN 38 ± 6  64 ± 15 42 ± 7  [CpG - 20 bases - Sup] ODN  7 ±4  48 ± 13 24 ± 8  [CpG - 20 bases - Cont] ODN * 94 ± 7 >100 ± 14  99 ±11 [Sup - CpG] ODN  0 ± 0  0 ± 0 0 ± 0 [Sup - 20 bases - CpG] ODN  8 ± 5 9 ± 3 2 ± 1 [CpG - Sup] ODN * + CpG ODN * >100 ± 16  >100 ± 15  98 ± 13[CpG - 5 bases - Sup] ODN * + >100 ± 18  >100 ± 11  98 ± 20 CpG ODN *[CpG - 10 bases - Sup] ODN + 58 ± 7 75 ± 9 66 ± 9  CpG ODN * [CpG - 20bases - Sup] ODN + 27 ± 5  26 ± 10 30 ± 8  CpG ODN * [Sup - CpG] ODN +CpG ODN *  9 ± 4 11 ± 4 8 ± 5 [Sup - 20 CT - CpG] ODN + CpG  5 ± 1  9 ±3 13 ± 2  ODN * BALB/c spleen cells were stimulated in vitro with 1 μMof each ODN, and the number of cells activated to secrete cytokinedetermined 8 hours later by ELIspot. The percent of cells activated tosecrete cytokine was calculated by the formula: (# of cells activated bytest ODN) − (background)/(# of cells activated by CpG ODN) −(background) × 100%. Two different control (ODN₁₄₇₁, and ODN₁₆₁₂), CpG(ODN₁₄₆₆ and ODN₁₅₅₅) and suppressive (ODN₁₅₀₂ and ODN_(H154)) ODN gavesimilar results in these experiments. Results represent the average of2-4 assays/data point. Table II shows typical numbers of cytokinesecreting cells/10⁶. * Stimulatory ODN, p < .05.

The trivial possibility that the CT spacer somehow reduced CpG activitywas eliminated by substituting a “control” motif for the 3′ suppressivemotif. The resulting ODNs were fully stimulatory (Table III).

The impact of placing a suppressive motif 5′ to a CpG motif was thenexamined. ODNs with a suppressive motif in the 5′ position inducedlittle or no immune activation, even when the CpG motif was shifted upto 20 bp downstream from the suppressive motif (Tables II and III). Thislack of activity could not be attributed to the 3′ location of the CpGmotif, since CpG ODNs with a ‘control’ sequence at the 5′ end wereimmunostimulatory. All ODNs containing a suppressive motif in the 5′position also inhibited co-administered CpG ODN (Tables II and III).These findings indicate that the relative position of stimulatory andsuppressive motifs determines the immunomodulatory properties of DNA.

Example 5 Suppressive ODNs Block the Development of Collagen-InducedArthritis

This example demonstrates the ability of suppressive ODNs to block thedevelopment of collagen-induced arthritis (an animal model of rheumatoidarthritis).

DBA/1 LacJ mice provide a murine model of human inflammation. Ten totwenty DBA/1 LacJ mice per group were injected with type II collagen incomplete Freund's adjuvant (CII/CFA) on day 0, and with type II collagenin incomplete Freund's adjuvant (CII/IFA) on day 21 to induce arthritis(see FIG. 17). The study groups included animals treated with 200 μg ofsuppressive ODN A151 (SEQ ID NO: 2), control ODN 1612 (SEQ ID NO: 29) orPBS on days −3, 0, 3, 7, 10, 14, 18 and 21. The incidence of arthritisand clinical score were monitored twice weekly. Antigen-specific humoraland cellular immune responses, and local expression of pro-inflammatorycytokines, were also investigated. Table IV shows the incidence ofarthritis and clinical score on day 56 of treatment. Treatment with ODNA151 (SEQ ID NO: 2) significantly reduced both the percentage of micethat developed arthritis (FIG. 18A), and the arthritis clinical score(FIG. 18B).

TABLE IV Incidence of arthritis and clinical score. Incidence (%)Clinical Score PBS-control 95 (19/20) 7.2 +/− 0.7 ODN 1612-treated 90(9/10) 5.9 +/− 1.1 A151-treated 50 (10/20) 1.7 +/− .04* Data representresults from day 56. *.p < 0.05, as compared with PBS-control or ODN1612-treated mice.

In addition, production of anti-CII antibody was suppressed by A151treatment. In FIG. 19, sera were collected on day 35 and assayed forIFNγ by ELISA assay, as described above. Standard curves wereconstructed by serial dilutions of a mixture of sera from arthriticmice. In FIG. 20, spleen cells from naive, PBS or A 151-treated micewere isolated on day 22 (FIG. 20A) or 35 (FIG. 20B). Cells werestimulated in vitro with 50 μg/ml of CII for 72 hours and culturesupernatants were assayed for IFN-gamma detection.

In vivo (local) expression of proinflammatory cytokine was alsosuppressed by treatment with ODN A151 (FIG. 21). The hind paws oftreated animals were removed on day 35. Total RNA was extracted fromtissue homogenates, and mRNA of the pro-inflammatory cytokine IL-1 β wasmonitored by RT-PCR.

While this disclosure has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments may be used and it isintended that the disclosure may be practiced otherwise than asspecifically described herein. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims:

1. A method of treating or preventing an inflammatory arthropathy in asubject comprising: administering to a subject having or at risk ofdeveloping the inflammatory arthropathy a therapeutically effectiveamount of an oligodeoxynucleotide comprising the oligodeoxynucleotidesequence set forth as SEQ ID NO: 1, thereby suppressing an immuneresponse in the subject and treating or preventing the inflammatoryarthropathy in the subject.
 2. The method of claim 1, wherein theoligodeoxynucleotide is administered topically, parenterally, orally,intravenously, intra-muscularly, sub-cutaneously, or intra-articularly.3. The method of claim 1, wherein the oligodeoxynucleotide is from 16 to100 nucleotides in length.
 4. The method of claim 1, wherein theoligodeoxynucleotide is from 16 to 30 nucleotides in length.
 5. Themethod of claim 1, further comprising administering an additionalanti-inflammatory, immunosuppressive, or anti-arthritis agent.
 6. Themethod of claim 5, wherein the agent is a biological response modifier,a disease-modifying antirheumatic drug, a steroid, a nonsteroidalanti-inflammatory drug, or a Cyclo-Oxygenase-2 inhibitor.
 7. The methodof claim 5, wherein the agent is anakinra, etanercept, infliximab,leflunomide, prednisone, cortisone, celecoxib, choline magnesiumtrisalicylate, diclofenac, diclofenac potassium, diclofenac XR,diflunisal, etodolac, etodolac ER, fenoprofen, flurbiprofen oral,ibuprofen, indomethacin, indomethacin SR, indomethacin suppositories,ketoprofen, ketoprofen ER, meclofenamate, meloxicam, nabumetone,naproxen, naproxen CR, naproxen ER, oxaprozin, piroxicam, rofecoxib,salsalate, sulindac, or tolmetin sodium, hyaluronan, or hylan G-F20. 8.The method of claim 1, wherein the oligodeoxynucleotide consists of thenucleic acid sequence set forth as SEQ ID NO:
 1. 9. The method of claim1, wherein the oligodeoxynucleotide is administered systemically.
 10. Amethod of treating inflammatory arthropathy in a subject comprisingcontacting immune cells with a therapeutically effective amount of anoligodeoxynucleotide of 16 to 30 nucleotides in length, wherein theoligodeoxynucleotide comprises the nucleotide sequence set forth as SEQID NO: 1 and transferring the immune cells to a subject having or atrisk of developing inflammatory arthropathy, thereby treating theinflammatory arthropathy.
 11. The method of claim 10, wherein the immunecells are transplanted.
 12. The method of claim 10, wherein the immunecells are transferred intravenously.
 13. The method of claim 10, whereinthe oligodeoxynucleotide consists of the nucleic acid sequence set forthas SEQ ID NO: 1.